Polysulfides compounds and lipid peroxidation inhibitor containing the polysulfide compounds as active ingredient

ABSTRACT

A lipid peroxidation inhibitor contains a polysulfide compound represented by formula (I) as an active ingredient: 
     
         R.sup.1 --(S).sub.m --R.sup.2                              (I) 
    
     wherein R 1  and R 2  are same or different hydrocarbon chains having 1 to 16 carbon atoms and m is an integer of 3 to 10. Among compounds (I) are found novel which are: 
     
         R.sup.3 --(S).sub.n --R.sup.4                              (II) 
    
     wherein R 3  and R 4  are same or different lower alkenyl groups and n is an integer of 6 to 10. Pharmaceutical compositions which contain a polysulfide compound represented by formula (I) as an active ingredient, for preventing and curing diseases of thrombosis, arteiosclerosis, liver troubles, pulmonary edema, skin troubles, eye troubles and aging are also disclosed.

This is a continuation of application Ser. No. 07/718,763 U.S. Pat. No.5,149,879, filed on Jun. 21, 1991.

BACKGROUND OF THE INVENTION

i) Field of the Invention:

The present invention relates to polysulfide compounds and a lipidperoxidation inhibitor containing the polysulfide compounds as an activeingredient.

ii) Description of the Background Art:

It is well known that a variety of vital phenomena involve a lipidperoxidation reaction, which is initiated by a free radical reaction. Inparticular, if the chain reaction of free radical is not cut off, anoxygen radical, a so-called an active oxygen produced in any creatureinduces lipid peroxidation against lipids present as a basic material ofa vital organism, for example, against polyvalent fatty acidsconstituting a matrix of a biomembrane.

That is, a lipid free radical and a peroxy free radical are produced ina series of steps to form a harmful peroxide lipid against the vitalorganism.

Hence, the lipid peroxidation reaction is concerned in a number ofsicknesses or diseases, for instance, hemolytic anemia, variousinflammations, myocardial infarction, cirrhosis of the liver,atherosclerosis and so forth, and further it is considered that thisreaction participates carcinogenesis and aging.

Thus, for preventing and curing sicknesses and diseases due to suchlipid peroxidation, development of drugs or pharmaceuticals forinhibiting the lipid peroxidation has been demanded. Some drugs forinhibiting the lipid peroxidation have been proposed, but cannotnecessarily have sufficient efficiency.

Under the above circumstances, the present inventors have carried outresearches for developing drugs or pharmaceuticals having a clinicallyexcellent lipid peroxidation inhibiting effect and have found that afirst group of polysulfide compounds having a formula (I):

    R.sup.1 --(S).sub.m --R.sup.2                              (I)

wherein R¹ and R² are same or different hydrocarbon chains having 1 to16 carbon atoms and m is an integer of 3 to 10, which are obtained by anextraction from the Allium plants widely used as foods and drugs orpharmaceutical materials particularly garlic, scallion, elephant garlicor the like or a treated material thereof or by a chemical synthesis,possess an excellent lipid peroxidation inhibiting effect, and that asecond group of polysulfide compounds included in the first group of thepolysulfide compounds, having a formula (II):

    R.sup.3 --(S).sub.n --R.sup.4                              (II)

wherein R³ and R⁴ are same or different lower alkenyl groups and n is aninteger of 6 to 10, are novel compounds.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide a lipidperoxidation inhibitor containing polysulfide compounds represented bythe following forumula (I) as an active ingredient:

    R.sup.1 --(S).sub.m --R.sup.2                              (I)

wherein R¹ and R² are same or different hydrocarbon chains having 1 to16 carbon atoms and m is an integer of 3 to 10.

It is another object of the present invention to provide polysulfidecompounds represented by the following formula (II):

    R.sup.3 --(S).sub.n --R.sup.4                              (II)

wherein R³ and R⁴ are same or different lower alkenyl groups and n is aninteger of 6 to 10.

It is still another object of the present invention to provide apharmaceutical composition containing polysulfide compounds representedby formula (I) as an active ingredient, for preventing and curingdiseases of thrombosis, arteiosclerosis, liver troubles, pulmonaryedema, skin troubles, eye troubles and aging.

The above and other objects, features and advantages of the presentinvention will more fully appear from the following description of thepreferred embodiments with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation showing lipid peroxidationinhibiting effect by the ratio between thiobarbituric acid-reactivesubstances TBA-RS(S) and TBA-RS(C) of polysulfides of the presentinvention as index;

FIG. 2 is a graphical representation showing lipid peroxidationinhibiting effect by the ratio between the thiobarbituric acid-reactivesubstances TBA-RS(S) and TBA-RS(C) with reference to sampleconcentrations of 1 to 25 μg/ml;

FIG. 3 is a graphical representation showing lipid peroxidationinhibiting effect by a ratio between fluorescence intensities F.I.(S)and F.I.(C) of polysulfides of the present invention as index; and

FIG. 4 is a graphical representation showing lipid peroxidationinhibiting effect by the ratio between the fluorescence intensitiesF.I.(S) and F.I.(C) of polysulfides of with reference to sampleconcentrations of 1 to 25 μg/ml.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

As to the hydrocarbon chains represented by R¹ and R² in formula (I),linear, branched or cyclic alkyl, alkenyl or alkynyl groups are given,and, in particular, lower alkyl, alkenyl or alkynyl groups arepreferable. As examples of the lower alkyl groups, alkyl groups having 1to 6 carbon atoms such as methyl, ethyl, n-propyl, hexyl, iso-propyl,tert.-butyl, cyclohexyl and the like are given. As examples of the loweralkenyl groups, alkenyl groups having 2 to 5 carbon atoms such as vinyl,allyl, 1-propenyl, iso-propenyl, 1,3-butadienyl and the like are given.As examples of the lower alkynyl groups, alkynyl groups having 2 to 5carbon atoms such as 2-propynyl, 2-butynyl, 2-pentene-4-ynyl and thelike are given. As to the lower alkenyl groups represented by R³ and R⁴in general formula (II), the similar groups to the above-describedalkenyl groups for R¹ and R² are given.

In formula (I), m is an integer of 3 to 10, and 3 to 8 are morepreferable. In formula (II), n is an integer of 6 to 10, and 6 to 8 aremore preferable.

As regards preferable examples of the compounds represented by formula(I), diallyl trisulfide, diallyl tetrasulfide, diallyl pentasulfide,diallyl hexasulfide, diallyl heptasulfide, diallyl octasulfide,methylallyl trisulfide, methylallyl tetrasulfide, methylallylpentasulfide, methylallyl hexasulfide, methylallyl heptasulfide,di(1-propenyl) trisulfide, di(1-propenyl) tetrasulfide, di(1-propenyl)pentasulfide, di(1-propenyl) hexasulfide, di(tert.-butyl) tetrasulfide,di(tert.-butyl) pentasulfide, allylvinyl tetrasulfide, allyl tert.-butyltetrasulfide and the like are given.

As to preferable examples of the compounds represented by generalformula (II), diallyl hexasulfide, diallyl heptasulfide, diallyloctasulfide, allylvinyl hexasulfide, allyl 1-propenyl hexasulfide andthe like are given.

The polysulfide compounds represented by formula (I), to be used in thepresent invention, can be produced by either an extraction from theAllium plants or a chemical synthesis.

The Allium plants to be used for the extraction are plants included inthe Allium genus of the Liliaceae family, and more specifically, garlic(Allium sativum), scallion (Allium chinense or Allium Bakeri Regel),elephant garlic (Allium ampeloprasum) and the like.

The extraction can be carried out from the whole plant, preferably, frombulbs containing divided semispherical or bowllike. Also, a cell lumpobtained by a tissue culture or other methods, a callus, a regeneratedplant can be used.

The extraction method is not restricted, and this treatment may becarried out by any conventional techniques commonly used in the exactionof crude drugs. That is, the extraction can be carried out by immersingthe plant in water or an organic solvent mixible with the water.Regarding the organic solvent used as an extraction treating solvent,lower alcohols, for example, monovalent alcohols having 1 to 3 carbonatoms can be used, and particularly ethanol is preferable. In theextraction, as a matter of course, a crushed or disintegrated plant ispreferably used in order to improve the efficiency.

The polysulfide compounds represented by formula (I) are nonpolarmaterials produced in the extraction and are not only contained in theextracted solution but also adsorbed by the residue. The extraction ofthe polysulfide compounds from the residue can be carried out byimmersing the residue in an organic solvent including or excluding thewater or by the steam distillation. In this case, as examples of theorganic solvent to be used as an extractant, lower alcohols, acetone andthe like as a solvent mixible with the water and chloroform, ethylacetate, hexane and the like as a solvent unmixible with the water aregiven. In these solvents, particularly preferable solvents are ethylacetate, chloroform and hexane.

The extractant is removed from the obtained extracted solution bydistillation, or the water is added to the extracted solution so as toextract with an organic solvent capable of partitioning the water toobtain the extract. The extract is appropriately separated by thechromatography using a normal phase adsorbent (preferably silica gel) ora reversed phase adsorbent to obtain polysulfide compounds.

On the other hand, a production of polysulfide compounds by chemicalsynthesis is performed by reacting, for example, diallyl disulfide withcupric chloride-ferric chloride and the like as a catalyst at roomtemperature, as disclosed in "Organic Sulfur Chemistry" ("Volume:Synthetic Reaction"), p. 98, by Shigeru OAE, Kagaku Dojin, 1982.

The polysulfide compounds represented by formula (I), obtained asdescribed above, exhibit an excellent lipid peroxidation inhibitingeffect within tissue of a liver microsome, blood or the like. Further,since these compounds are the extract naturally occurring from the plantsuch as garlic or the like or analogous plants, the safety is high.Hence, the polysulfide compounds represented by formula (I) as a lipidperoxidation inhibitor can be utilized for prevention or cure of avariety of diseases or sicknesses such as thrombosis, arteiosclerosis,liver trouble, pulmonary edema, skin trouble, eye trouble and aging.

According to the present invention, a lipid peroxidation inhibitorcontains at least one polysulfide compound represented by formula (I) asan active ingredient. The lipid peroxidation inhibitor can be mixed witha liquid or solid pharmaceutically acceptable preparational auxiliarycomponents or carriers such as excipients, binders, diluents and thelike to form any drug form such as powders, granules, tablets, capsules,liquids and injections, and can be administrated orally or parenterally.

The dose of the lipid peroxidation inhibitor is properly varieddepending upon age, weight, symptom and so forth. The oral dose foradult in terms of polysulfide compounds represented by formula (I) ispreferably 1 mg to 10 g per day, which can be administrated in one timeor divided into several times. As a matter of course, when the lipidperoxidation inhibitor is administrated, it can be mixed with anotherdrug, as required.

Then, the examplary embodiments of the present invention will now bedescribed in detail, and it should be understood that these embodimentsare given for illustration of the invention and are not intended to belimitative therefor.

EXAMPLE 1

Production of diallyl polysulfide compounds from garlic:

10 kg of garlic was crushed and extracted with 10 l of 20% ethanol atroom temperature. After the solid was separated from the solution, theextracted solution was concentrated under reduced pressure, and waterwas added to the solution. The extraction was carried out by 5 l ofethyl acetate. On the other hand, the residue was extracted by 5 l ofethyl acetate. The obtained extracted solution was mixed with thepreviously obtained ethyl acetate extracted solution, and the mixedextracted solution was concentrated under reduced pressure to obtain anextract. The obtained extract was treated by a silica gel columnchromatography with n-hexane as a solvent. The obtained sulfide fractionwas separated by high performance liquid chromatography (TSK gel ODS80TM (TOSOH), 90% ethanol eluate) to obtain 81 mg of diallyl trisulfide,154 mg of diallyl tetrasulfide, 102 mg of diallyl pentasulfide, 48 mg ofdiallyl hexasulfide and 11 mg of diallyl heptasulfide.

Data of the obtained polysulfides are shown as follows:

(1) Diallyl trisulfide:

1) External appearance: slightly yellow oil

2) ¹ H-NMR (deuterochloroform) δ: 3.51(2H, d,J=7.0 Hz), 5.2-5.3(2H,m),5.8-6.0(1H,m)

3) ¹³ C-NMR (deuterochloroform) δ: 41.7, 119.1, 132.7

4) High Resolution Mass Spectra: as C₆ H₁₀ S₃ Theoretical value:177.995; Measured Value: 177.997

(2) Diallyl tetrasulfide:

1) External appearance: slightly yellow oil

2) ¹ H-NMR (deuterochloroform) δ: 3.59(2H,d,J=7.3 Hz), 5.2-5.3(2H,m),5.8-6.0(1H,m)

¹³ C-NMR (deuterochloroform) δ: 42.1, 119.5, 132.5

4) High Resolution Mass Spectra: as C₆ H₁₀ S₄ Theoretical value:209.962; Measured Value: 209.964

(3) Diallyl pentasulfide:

1) External appearance: slightly yellow oil

2) ¹ H-NMR (deuterochloroform) δ: 3.61(2H, d, J=7.3 Hz), 5.2-5.3(2H, m),5.8-6.0(1H, m)

3) ¹³ C-NMR (deuterochloroform) δ: 42.6, 119.8, 132.2

4) High Resolution Mass Spectra: as C₆ H₁₀ S₅ Theoretical value:241.938; Measured Value: 241.938

(4) Diallyl hexasulfide:

1) External appearance: slightly yellow oil

2) ¹ H-NMR (deuterochloroform) δ: 3.62(2H, d, J=7.3 Hz), 5.2-5.3(2H, m),5.8-6.0(1H, m)

3) ¹³ C-NMR (deuterochloroform) δ: 42.5, 120.0, 132.2

4) High Resolution Mass Spectra: as C₆ H₁₀ S₆ Theoretical value:273.915; Measured Value: 273.913

(5) Diallyl heptasulfide:

1) External appearance: slightly yellow oil

2) ¹ H-NMR (deuterochloroform) δ: 3.62(2H, d, J=7.3 Hz), 5.2-5.3(2H, m),5.8-6.0(1H, m)

3) ¹³ C-NMR (deuterochloroform) δ: 42.5, 120.0, 132.2

4) High Resolution Mass Spectra: as C₆ H₁₀ S₇ Theoretical value:305.885; Measured Value: 305.884

EXAMPLE 2

Production of diallyl polysulfide compounds from diallyl disulfide:

55 g of anhydrous ferric chloride was dissolved in 400 ml of ether, andin this ethyl ether solution was added a solution where 11.5 g ofanhydrous cupric chloride was dissolved in 400 ml of acetonitrile. Inthis mixture, 25 g of diallyl disulfide was added, and stirred overnightin a stream of nitrogen at room temperature while the light wasshielded. The reaction mixture was poured into 400 ml of saturatedsodium chloride aqueous solution, and extracted four times by 400 ml ofn-hexane. Then, the obtained n-hexane layer dehydrated by anhydroussodium sulfate, and was then concentrated under reduced pressure. Theobtained sulfide fraction was separated by high performance liquidchromatography (TSK gel ODS 80TM (TOSOH), 90% ethanol eluate) to obtain90 mg of diallyl trisulfide, 81 mg of diallyl tetrasulfide, 75 mg ofdiallyl pentasulfide, 46 mg of diallyl hexasulfide and 36 mg of diallylheptasulfide.

TEST EXAMPLE 1

Lipid peroxidation inhibiting effect:

1) Test method:

Lipid peroxidation inhibiting effect of the polysulfide compoundsrepresented by formula (I) in liver microsomes was examined by using theformation of thiobarbituric acid-reactive substances (TBA-RS) andfluorescent substances as index.

That is, liver microsomes (see F. Itoh et al., Arch. Biochem. Biophys.264, 184(1988)) prepared from male Wistar rats were incubated for 5minutes at 37° C. in the presence or absence of the polysulfidecompound, and then 0.1 mM of ascorbic acid and 5 μM of ferrous sulfatewere added to the microsome suspension to initiate lipid peroxidation.The mixture was incubated for 3 hours at 37° C. The reaction wasterminated by adding 1 mM of EDTA.

i) Assay of thiobarbituric acid-reactive substances (TBA-RS):

An assay of malondialdehyde produced by the lipid peroxidation wascarried out by the method of Buege et al. (Methods Bnzymol., 52,302(1978)).

ii) Measurement of fluorescent substances:

After the termination of the lipid peroxidation, the microsomes wereseparated by Sephadex G-25 column chromatography by using 0.1M of sodiumphosphate buffer solution containing 1 mM of EDTA. The fluorescenceemitted by the obtained peroxidized microsome was measured by the directmethod (see F. Itoh et al., Arch. Biochem. Biophys., 264, 184 (1988)).

2) Results:

i) The formation of the TBA-RS as the index of lipid peroxidation wasdetermined in the presence and absence of the polysulfide compound toobtain TBA-RS(S) and TBA-RS(C), respectively. A sample concentration wasdetermined to be 25 μg/ml, and a ratio TBA-RS(S)/TBA-RS(C) between theTBA-RS(S) and TBA-RS(C) was obtained. As a result, a TBA-RS inhibitingeffect was observed in the polysulfide compounds, as shown in FIG. 1.Further, when the sample concentration was varied to 1, 5, 10, 15 and 25μg/ml, the ratio TBA-RS(S)/TBA-RS(C) for each compound was obtained, andas a result, a concentration dependence was clearly observed, as shownin FIG. 2.

ii) It is well known that with the lipid peroxidation, fluorescentsubstances were produced. By using the formation of fluorescentsubstances as the index, a ratio F.I.(S)/F.I.(C) between fluorescentintensities F.I.(S) and F.I.(C) was obtained in the presence and absenceof the polysulfide compound. As a result, it was observed that thepolysulfide compounds inhibit the formation of the fluorescentsubstances, as shown in FIG. 3. Further, when the sample concentrationwas varied to 1, 5, 10, 15 and 25 μg/ml, the fluorescent intensity ratioF.I.(S)/F.I.(C) for each compound was obtained, and as a result, aconcentration dependence was clearly observed, as shown in FIG. 4.

As described above, it was observed that the polysulfide compounds havea remarkable lipid peroxidation inhibiting effect.

According to the present invention, lipid peroxidation inhibitorscontaining the polysulfide compounds represented by formula (I) as anactive ingredient are effective for the prevention and cure of a varietyof diseases or sicknesses due to lipid peroxidation, which include,thrombosis, arteiosclerosis, liver troubles, pulmonary edema, skintroubles, eye troubles and aging.

Although the present invention has been described in its preferredembodiments, it is readily understood that the present invention is notrestricted to the preferred embodiments and that various changes andmodifications thereof can be made by those skilled in the art withoutdeparting from the spirit and scope of the present invention.

What is claimed is:
 1. A method of inhibiting lipid peroxidation in apatient in need thereof comprising administering a lipid peroxidationinhibiting effective amount of a lipid peroxidation inhibitorcomprising:a polysulfide compound of formula (I) as the activeingredient

    R.sup.1 --(S).sub.m --R.sup.2                              (I)

wherein R¹ and R² are the same or different hydrocarbon chains having 1to 16 carbon atoms and m is an integer of 3 to 10; and apharmaceutically acceptable carrier.
 2. The method of claim 1, whereinR¹ and R² are the same or a different lower alkenyl groups and m is aninteger of 3 to 10.